Malaria constitutes a permanent catastrophe. Annually, the disease kills between 1 and 2 million Africans and the economic losses due to malaria constitute a hindrance for economic development. In areas of stable malaria transmission the disease mainly affects children, because adults have acquired immunity which protects them against severe malaria syndromes and most febrile malaria episodes. However, pregnant women constitute an important exception to this rule since they often suffer from severe malaria attacks.
Further, even in the absence of overt clinical symptoms the presence of parasites in pregnant women can have very serious consequences for both mother and child because the infection cause maternal anaemia, as well as premature delivery, low birth weight, and increased infant mortality (Brabin, 1983).
Thus, pregnancy-associated malaria (PAM) is a major health problem in malaria-endemic areas and on a world basis it affects millions of pregnant women and their offspring. In endemic areas, PAM is concentrated among primigravid women, indicating that protective immunity to PAM is acquired as a function of parity and that it is possible to make a vaccine protecting against PAM.
Malaria is caused by unicellular parasites living and multiplying asexually in the red blood cells (RBC). In each 48-hour cycle, the parasites invade RBC, multiply within them, and eventually burst them, before they go on to invade new RBC. Four Plasmodium species cause human disease, but by far the most of the malaria disease burden is caused by Plasmodium falciparum, which is also the cause of PAM.
RBC infected by the late developmental stages of P. falciparum blood parasites are not found in the peripheral circulation, as they adhere to receptors on the endothelial lining. This adhesion, called sequestration, is mediated through parasite-encoded, clonally variant surface antigens (VSA) inserted into the membrane of the infected RBC (IRBC) and is thought to be an immune evasion strategy, possibly evolved to avoid splenic clearance.
The best-characterised VSA are encoded by the var genes. This gene family, encompassing about 60 members per genome, encodes the variant protein P. falciparum erythrocyte membrane protein 1 (PfEMP1), which is located on the surface of the P. falciparum-infected erythrocytes where it mediates adhesion.
A given parasite expresses only one PfEMP1 at a time, but in each generation a fraction of the daughter parasites may switch to expression of alternative PfEMP1 species through an unknown process. Different PfEMP1 molecules have different receptor specificities, and clonal switching between expression of the various var gene products in a mutually exclusive manner allows the parasite to modify its adhesion properties, which in turn determines in which tissue the parasite can sequester (Wahlgren et al., 1999).
PAM is caused by the accumulation of parasites in the intervillous space of the placenta, where parasites adhere to the syncytlotrophoblast.
The glycosaminoglycan chondroltin sulphate A (CSA) can mediate parasite adhesion in vitro, and although CSA-adhering parasites are rarely found in non-pregnant hosts, placental parasites preferentially or perhaps even exclusively bind to CSA, whereas they seldom bind to CD36, which is the most common sequestration ligand for parasites from non-pregnant hosts. Thus, it seems that the placenta constitutes a niche for antigenically distinct parasite variants that have evolved to sequester exclusively at this site.
According to this theory, such parasites cannot survive in non-pregnant hosts. Primigravid women in endemic countries are consequently fully susceptible to CSA-adhering parasites, even if they have acquired protection to most other parasite variants. With increasing parity, an increasing proportion of women has encountered such parasites during previous pregnancies and produced protective antibodies against them, which in turn explains the parity-dependency of susceptibility to PAM. This notion is supported by the fact that plasma from some pregnant women can block the binding of placental parasites to CSA and that the proportion of pregnant women with plasma that block binding at partum increases with parity (Duffy and Fried, 1999).
As PAM can occur even in women who have acquired immunity to malaria, the parasites causing PAM must be able to escape the immunological effector mechanisms that control parasite multiplication in immune hosts. This is supported by the fact that VSA expressed by parasites isolated from the placenta of women with PAM are not recognised by plasma antibodies from clinically immune adult males or women who have not been pregnant, implying that the VSA expressed by parasites causing PAM cannot multiply successfully in men, but only in women harbouring a placenta.
Another significant characteristic shared by VSA expressed by placental parasite isolates is that the levels of plasma IgG in malaria-exposed pregnant women are positively correlated to parity (parity-dependent IgG recognition). Together, these observations suggest that parasites causing PAM express VSA molecules that do not cross-react serologically with the VSA expressed on parasites which do not sequester in the placenta, and that a vaccine protecting against PAM, should induce antibodies which recognise VSA on placental parasites but not VSA expressed by parasites isolated from peripheral blood of men or non-pregnant women.
The ability of plasma to block the binding of placental parasites to CSA which are found in some malaria-exposed pregnant women, is independent of the geographic origin of plasma as well as parasites (Fried et al., 1998). These data suggest that the VSA responsible for placental adhesion to CSA are not only functionally and antigenically distinct from other molecules present at the IRBC surface, but also that they share relatively conserved antigenic determinants. The fact that many women in areas of low malaria transmission intensity suffer from PAM indicates that even though parasites in the peripheral blood of non-pregnant individuals do not express the protein responsible for placental adhesion, most parasite genomes carry genes encoding the protein, which can be selected for or actively turned on if the parasite infects a pregnant women. Together these data indicate that the gene encoding the protein responsible for PAM is carried by most parasites, and that it is conserved and structurally different from other VSA.
Parasites isolated from peripheral blood of non-pregnant individuals do not normally bind to CSA in vitro but after several rounds of in vitro panning on CSA bound to plastic, parasite lines that bind specifically to CSA can be established. These parasite lines normally express VSA with several phenotypical features similar to VSA expressed by placental parasites: i) CSA-selected parasites bind to placental tissue, ii) CSA-selected parasites are recognised by plasma in a gender- and parity-dependent manner (Staalsoe et al., 2001), iii) plasma from pregnant women often block the adhesion of CSA-selected parasites to both CSA and placental tissue, iv) CSA selected parasites do not bind CD36. None of these characteristics are normally found in the parental parasite lines before CSA selection. Thus, in vitro-generated, CSA-binding parasite lines resemble placental parasites, and comparison of gene expression between CSA-binding parasite lines and the parental line can be( used as a tool to identify the gene(s) involved in the pathogenesis of PAM.
Several groups of researchers have identified specific PfEMP1 molecules that can mediate binding to CSA (Buffet et al., 1999). One such molecule, FCR3.varCSA, has been cloned recently and its prophylactic and therapeutic applicability with respect to PAM has been claimed (Scherf et al. WO 01/16326). However, this parasite isolate was not shown to be gender-specifically recognised by immune sera. One must keep in mind, however, that the structure of PfEMP1 has been optimised during evolution to mediate binding to different ligands.
Since CSA bearing proteins also exist on the endothelial surface outside the placenta, and CSA is notoriously a sticky molecule, the demonstration that a species of PfEMP1 mediates binding to CSA does not in itself constitute evidence that the molecule mediates binding to placenta in vivo and is involved in the pathogenesis of PAM. As for the FCR3.varCSA, it has subsequently been reported that a FCR3CSA strain with a FCR3varCSA knockout is still able to adhere in vitro to CSA.
The present invention relates to a particular PfEMP1, VAR2CSA and the var2csa gene, which serves a unique function for Plasmodium falciparum. 
WO 00 25728 describes the DNA and protein sequences derived from the sequencing of chromosome 2 of Plasmodium falciparum 3D7. The sequences are disclosed for use in a vaccine against malaria. This publication does not address any expression characteristics, binding abilities or antigenic properties for any of the disclosed sequences, nor does this application does not relate to pregnancy associated malaria. Furthermore, the chromosome 2 of P. falciparum contains numerous fragmented and truncated var sequences.
Sequence ID No 3 is the protein sequence of the DNA in sequence in SEQ ID No 213. This sequence only disclose a fragment of 1323 bp/440 aa coding for a truncated PfEMP1 encoding only the conserved exon2 part of the molecule.
In the EMBL online database, Database accession no. AE014844, disclose Plasmodium falciparum 3D7 chromosome 12 section 1 of 9 of the complete sequence.
This sequence is identical to var2csa and is derived from the sequencing of the whole Plasmodium falciparum 3D7 genome. The online reference is merely a sequence submisson and does not contain any information on function of sequence nor any relevance for a malaria vaccine or a pregnancy associated malaria vaccine.
In the present application, the var2csa sequence is provided as SEQ ID NO.: 1 and is excluded from the embodiments pertaining to the nucleic acid sequences as such. An open reading frame (ORF) comprises nucleic acids No. 48802-56805. This ORF is the translation of the nucleotide sequence of var2csa derived from the sequencing of the whole Plasmodium falciparum 3D7 genome. In the present application, this protein sequence is provided as SEQ ID NO.: 2 and is excluded from the embodiments relating to amino acid sequences as such.
In the EMBL online database, Database accession no. BQ739499 (PfESToab46 g01.y1) describes a cDNA fragment of 548 bp identical to var2csa. This fragment is derived from sequencing of a Plasmodium falciparum 3D7 EST library. This online submisson does not contain any information on this sequence in relation to a vaccine against malaria.
WO 01/16326 disclose a PfEMP1 sequence for the use in a vaccine against PAM and for the use of treatment of PAM. The sequence FCR3varCSA is from the Plasmodium falciparum strain FCR3 and is fundamentally different from var2csa in spite of the proteins belonging to the same variant surface antigen family (var).
As mentioned above, PfEMP1 genes show both intra and inter genomic variation, and the global repertoire of PfEMP1 proteins is assumed to be very large. The common features shared by the PfEMP1 family of genes and proteins (Smith et al., 1995) are the organisation of the genes (two exons and an intron), and the presence of domain structures that can be classified as Duffy Binding ligand-like (DBL) or cysteine-rich interdomain region (CIDR)(Smith et al., 2000).
In addition the proteins share a relative conserved c-terminal tail consisting of a trans membrane region and a relatively short intracellular domain. However, it must be stressed that the genes and the encoded proteins vary considerably between each other; both with regards to sequence (primary structure) and organisation of the domains (Lavstsen et al., 2003).
It is also clear that expression of different PfEMP1 molecules confer parasite different functional (Smith et al., 1995). (Smith et al., 2000; Robinson et al., 2003) and antigenic characteristics (Salanti et al., 2003). Furthermore, it is obvious that an efficient PfEMP1-based vaccine against malaria and PAM in particular is limited to a few specific PfEMP1 types.
Within PfEMP1 domains classified as belonging to the same group and subgroup (i.e. DBLα, DBLβ, CIDRγetc) short identity blocks of 2-14 amino acids can be identified between hyper variable blocks of varying lengths (of up to several hundred amino acids) in which there is no or very little homology between randomly chosen PfEMP1s.
Although the sequence of the entire P. falciparum genome is known, the VAR2CSA protein and its role in the pathogenesis of malaria has not previously been described. Accordingly, the present invention provides a new PfEMP1 molecule. The PfEMP1 molecules constitute a very large and diverse family of proteins, the prior identification of other PfEMP1 molecule does not suggest any function of VAR2CSA for the parasite, which is unique, and distinct from that of previously described PfEMP1s.
The domain structure of FCR3varCSA is somehow classic consisting of a “conserved” domain headstructure (DBL1α, CIDR1α, DBL2β), furthermore the CSA binding domain of this molecule has been mapped to the DBL3-γ of this molecule and until the discovery of VAR2CSA there was a general consensus about DBL3-γ as being the CSA binding domain.
The domain structure of VAR2CSA is fundamentally different from all other PFEMP1 proteins, including FCR3varCSA.
The first 3 domains do not fit any current classification and has been named DBLX—the last 3 domains are a unusual repetition of three ε domains: DBL1-X, DBL2-X, DBL3-X, DBL4ε, DBL5ε, DBL6ε.
What is most noteworthy is that VAR2CSA does not have the DBL-γ domain which was thought to be the domain mediating adhesion to CSA and to placenta nor is there any CIDR domains present.
A ClustalX alignment of the exon1 of the two proteins only gives an overall identity of 18.3% and it is not even possible to make a reasonable aligment from the nucleotide sequence. Thus the two proteins are very different in both primary sequence structure and in domain architectural structure.
Summa summarum, Plasmodium falciparum erythrocyte membrane protein-1 (PfEMP1), is a highly polymorphic and diverse family of proteins. Every parasite genome carries as mentioned about 60 genes encoding PfEMP1 and the repertoire of PfEMP1 genes differ from parasite genome to parasite genome. Thus, PfEMP1 genes show both intra and inter genomic variation, and the global repertoire of PfEMP1 proteins is assumed to be very large.